The Study Of Serum MicroRNA For Diagnosis And Chemotherapeutic Response In Patients With Non-small Cell Lung Cancer

PART I The study of serum micro RNA for diagnosis in patients with non-small cell lung cancerBackgroudLung cancer remains one of the major cancer diseases worldwide. Approximately 1.3 million patients with lung cancer die each year, making it the cancer with the highest death toll. 75-80% of all lung cancer cases are non-small-cell lung cancers(NSCLC). Because NSCLC does not usually become clinically apparent until it reaches an advanced stage, greater than 75% of lung cancers are diagnosed after the disease is already locally advanced or metastatic, making them miss the optimal timing of surgery. The lack of effective early diagnostic is the main causes of high mortality in NSCLC. Therefore, the identification of diagnostic could help clinicians to treat early for NSCLC patients. Circulating tumor markers are attractive for cancer screening since they are blood-based tests that are minimally-invasive, relatively low-cost and easily repeatable. Useful tumor markers are valuable for clinical diagnosis, prognostic evaluation and therapy monitoring. Serum micro RNAs are attractive candidates to be used as cancer biomarkers. micro RNAs are a class of naturally occurring small(18-25 nucleotides) noncoding RNAs in eukaryotic cells that can postranscriptionally silence protein expression by binding to complementary target gene transcripts. An increasing number of studies have shown that micro RNAs participate in tumorigenesis and progression of various types of cancer, including lung, breast, gastric and colorectal cancers and so on. Analysis of micro RNAs expression has a potential application value in cancer diagnosis, treatment, and prognosis prediction. Furthermore, it has been shown that micro RNAs are present in human serum or plasma in a remarkably stable form that is protected from endogenous RNase activity, which highlights the potential of circulating micro RNAs to act as stable blood-based markers for the diagnosis or prognosis of cancer.ObjectiveThe expression levels of circulating serum micro RNAs in NSCLC, patients with benign lung diseases and healthy controls were detected in the study. The objective of the study is to evaluate the clinical diagnosis significance of the serum micro RNAs level changes in NSCLC patients.Methods1. One hundred and twenty patients with NSCLC, referred to the Beijing Chest Hospital, Capital Medical University between August 2012 and October 2013, were enrolled in the study. All patients were pathologically diagnosed with NSCLC, and had never received previous treatment. The control subjects consisted of forty-five patients with benign lung diseases and forty-five healthy volunteers. Serum samples of all the subjects were collected and the total micro RNA was extracted from serum using the circulating mi RNA isolation kit.2. Micro RNA expression profiling was done in three patients of NSCLC and three healthy control subjects using circulating serum micro RNA One Array®. A panel of different expression serum mi RNAs were found between NSCLC patients and control subjects.3. First, the reverse transcription reaction was performed in mi RNA samples of all the subjects. Subsequently, the real-time quantitative PCR(q RT-PCR) was carried out to identify the different expression mi RNAs independently in 120 patients with NSCLC and 90 control subjects. mi RNA-103 was used as an internal reference for the normalization of mi RNA qRT-PCR data of all the serum mi RNA samples.4. To evaluate the diagnostic value(sensitivity and specificity) of circulating serum mi RNAs for NSCLC, receiver-operating characteristic(ROC) curves were constructed. The potential clinical diagnostic significance of serum mi RNAs for NSCLC was investigated by comparing with other serum tumor markers which were used as routine clinical tests, such as the carcinoma embryonic antigen(CEA), and so on.Results1. According to the results of serum micro RNA array profiling,a panel of 26 mi RNAs were upregulated in sera of patients with NSCLC, while 24 mi RNAs were deregulated. Subsequently, the q RT-PCR was carried out to identify the 10 candidate serum mi RNAs of different expression, including 5 upregulated mi RNAs(mi R-22、mi R-125b、mi R-197、mi R-19b、mi R-27b) and 5 downregulated mi RNAs(mi R-15b、mi R-16、mi R-25、mi R-205、mi R-155).2. The results of the q RT-PCR data showed that, the expression levels of the 10 candidated circulating serum mi RNAs were detected effectively. The expression levels of serum mi R-125 b was significantly increased in patients with NSCLC compared with healthy controls and patients with benign lung diseases, the difference was statistically significant(Z = 2.238, P = 0.025); The expression levels of serum mi R-22 was significantly increased in patients with NSCLC compared with healthy controls and patients with benign lung diseases, the difference was statistically significant(Z = 2.129, P = 0.035); The expression levels of serum mi R-15 b was significantly decreased in patients with NSCLC compared with healthy controls and patients with benign lung diseases, the difference was statistically significant(Z = 2.590, P = 0.010). The expression levels of serum mi R-125 b was found to be significantly higher in stages III and IV than those of stages I and II, the difference was statistically significant(Z = 2.891, P = 0.004); The expression levels of serum mi R-15 b was significantly lower in stages III and IV than those of stages I and II, the difference was statistically significant(Z = 2.081, P = 0.040).3. The sensitivity of serum mi R-22 in early stage NSCLC(I stage + II stage) was significantly higher than CEA(43.5% and 21.7%, respectively), the difference was statistically significant(χ2 = 4.946, P = 0.026). The sensitivity of serum mi R-15 b in early stage NSCLC was significantly higher than CEA(41.3% and 21.7%, respectively), the difference was statistically significant(χ2 = 4.079, P = 0.043). The sensitivity of serum mi R-125 b in advanced stage NSCLC(Ⅲ stage + Ⅳstage) was significantly higher than early stage NSCLC(58.1% and 30.4%, respectively), the difference was statistically significant(χ2 = 5.875, P = 0.015).4. ROC curve analysis showed that serum mi R-22, mi R-125 b and mi R-15 b were of significant diagnostic value for NSCLC, and the area under the ROC curve(AUC) were 0.725(95% CI = 0.623-0.827)、0.704(95% CI= 0.601-0.808)、0.619(95% CI = 0.536-0.702), respectively. The diagnostic value for NSCLC of serum mi R-22, mi R-125 b and mi R-15 b were higher than CEA(AUC = 0.594,95% CI = 0.482-0.707).Conclusion1. In the study, 10 different expression mi RNAs in serum were selected by micro RNA One Array. The validated results of the q RT-PCR tests showed that micro RNA One Array to be a simple and feasible method for selecting different expression mi RNAs in serum.2. The results of the study showed that serum mi R-125 b and mi R-22 were significantly upregulated, while serum mi R-15 b was significantly downregulated. These results indicated that serum mi RNAs which as tumor markers have certain potential clinical value for NSCLC diagnosis.3. The study found that the sensitivity of serum mi R-22 and mi R-15 b in early stage NSCLC(I + II stage) significantly higher than CEA and CK-3A9 which indicated that mi R-22 and mi R-15 b may be as the reference indicators for early diagnosis of NSCLC.4. Difference expression of serum mi RNAs might be involved in the pathogenesis of NSCLC. Detection of serum mi RNAs has certain clinical values for auxiliary diagnosis and early diagnosis of NSCLC.PART II The study of serum micro RNA for chemotherapeutic response in patients with non-small cell lung cancerBackgroudBecause non-small-cell lung cancer(NSCLC) patients are often detected accidentally, most of the patients of NSCLC are in the late stage and have not the optimal opportunity for surgery treatment. In these situations, the therapeutic options are limited to chemotherapy and radiotherapy. Chemotherapy is the mainstream method of treatment against advanced NSCLC. The practice proved that combined chemotherapy can improve the survival rate of advanced NSCLC. However, and the effective rate of first-line chemotherapy is only 30%. Clinical resistances to chemotherapeutic drugs may result in not only treatment failure, but also severe damage to immune system and loss of optimal timing of other therapy. Patients undergoing chemotherapy require monitoring to assess tumor progression. Typically, the best response to chemotherapy is achieved after the first 3-4 cycles. The efficacy of the chemotherapy applied is mostly determined by evaluations of solid tumor method. However, this method to evaluate the effective of chemotherapy for NSCLC not only needs a long time(42 days at least) by evaluating macroscopic alterations of the tumor volume or diameter, but also needs the expensive data of imaging. Evaluations of solid tumor method has inadequacies in daily practice settings as in the case of patients with pleural effusions, diffuse nodules, or tumors with poorly defined margins, and could lead to incorrect interpretation of tumor response. Thus, the development of easier and cheaper tools with which to monitor the effects of chemotherapy in patients with NSCLC are urgently needed. It would be useful to know earlier whether a tumor responds to treatment, so as to adapt the therapy to each NSCLC individual and change the regimen in time. Tumor markers have been extensively used for prognosis prediction and monitoring treatment in patients with NSCLC, such as CEA, NSE, Cyfra21-1 and so on. Serum micro RNAs as the new types of tumor markers, the study of serum micro RNAs about prognosis prediction and monitoring treatment in patients with NSCLC are starting. An increasing number of studies has shown that micro RNAs participate in tumorigenesis and progression of various types of cancer. Analysis of micro RNAs expression has a potential application value in cancer treatment, and prognosis prediction.ObjectiveThe expression levels of circulating serum micro RNAs in patients with advanced NSCLC(Ⅲ + Ⅳ stages) pre- and post-chemotherapy were detected in the study. The objective of the study was to evaluate the chemotherapeutic response significance of the serum micro RNAs level changes in NSCLC patients.Methods1. Seventy-four patients with advanced NSCLC, referred to the Beijing Chest Hospital, Capital Medical University between August 2012 and October 2013, were enrolled in the study. Serum samples for 5 ml of advanced NSCLC patients pre- and post-chemotherapy were collected and the total micro RNA was extracted from sera using the circulating mi RNA isolation kit.2. The reverse transcription reaction was performed in mi RNA samples of total 144 subjects. Subsequently, the real-time quantitative PCR(q RT-PCR) was carried out to test the different expression mi RNAs in patients with advanced NSCLC, which were selected in the study of part I, including mi R-125 b, mi R-22 and mi R-15 b. The mi R-103 was used as an internal reference for the normalization of mi RNA q RT-PCR data of 144 serum mi RNA samples.3. The evaluation of the chemotherapeutic response was based on the standards of Response Evaluation Criteria in Solid Tumors(RECIST), defined as complete remission(CR), partial remission(PR), stable disease(SD), and progressive disease(PD).4. The changes of expression levels of serum micro RNAs in patients with advanced NSCLC pre- and post-chemotherapy were analysed in the study. The potential clinical application significance of serum mi RNAs for chemotherapeutic response in NSCLC patients was investigated.Results1. Chemotherapeutic effects after 2 cycles were evaluated on the 74 patients with advanced NSCLC. According to the RECIST, all the patients were partitioned into 4 groups as following: 0 complete response(CR), 34(45.9%) partial response(PR), 33(44.6%) stable disease(SD) and 7(9.5%) progression disease(PD).2. To determine the correlation between serum micro RNAs levels and chemotherapy response, the micro RNAs levels between pre- and post-chemotherapy were analyzed in 74 advanced NSCLC patients who received 2 cycles of chemotherapy. Generally, serum level of mi R-125 b significantly decreased after chemotherapy, the difference was statistically significant(Z = 2.196, P = 0.028). The levels of mi R-15 b significantly increased after 2 cycles of chemotherapy, the difference was statistically significant(Z = 2.650, P = 0.008). While there were no change in the level of serum mi R-22, the difference was no statistically significant(P > 0.05).3. Compared with pre-chemotherapy, levels of serum mi R-125 b in advanced NSCLC patients of responders(CR + PR) were significantly decreased after 2 cycles of chemotherapy, the difference was statistically significant(Z = 2.128,P = 0.033); levels of serum mi R-15 b in advanced NSCLC patients of responders(CR + PR) were significantly increased, the difference was statistically significant(Z = 2.794,P = 0.005). While the levels of serum mi R-125 b and mi R-15 b were no changes in the advanced NSCLC patients of non-responders(SD + PD), the difference was no statistically significant(P > 0.05).4. The results of the q RT-PCR data showed that the effectiveness of chemotherapy was significantly associated with the serum levels of micro RNAs before treatment. Among the advanced NSCLC patients with high levels of serum mi R-125 b, 34.9% responded to chemotherapy with either CR or PR. While the advanced NSCLC patients with low levels of serum mi R-125 b, 51.6% exhibited a response to chemotherapy. The difference in response to chemotherapy between high and low serum mi R-125 b patients with advanced NSCLC was statistically significant(χ2 = 5.879, P = 0.015). Among the advanced NSCLC patients with low levels of serum mi R-15 b, 37.5% responded to chemotherapy with either CR or PR. While the NSCLC patients with high levels of serum mi R-15 b, 52.9% exhibited a response to chemotherapy. The difference in response to chemotherapy was statistically significant(χ2 = 4.795, P = 0.029).Conclusion1. The results of the study showed that the expression levels of serum mi R-125 b and mi R-15 b in patients with advanced NSCLC were significantly changed pre- and post-chemotherapy, and the changes were associated with chemotherapeutic response. These results indicated that detecting the serum expression levels of mi RNAs have certain potential clinical value for chemotherapeutic response in advanced NSCLC.2. The levels of serum mi RNAs pre-chemotherapy in patients with advanced NSCLC were associated with chemotherapeutic response. The conditions of prognosis for patients with advanced NSCLC received chemotherapy can be estimated according to the expression levels of serum mi R-125 b and mi R-15 b in patients before treatment.